Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Avermectin, candicidin, epothilone, erythromycin, FK-506, FK-520, narbomycin, oleandomycin, picromycin, rapamycin, spincoyn, tetracycline, and tylosin are examples of such compounds. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. Polyketides can be divided into macrocyclic/polyether-type compounds, biosynthetically encoded by type-1 polyketide synthases (PKSs), and into multicyclic, aromatic compounds, whose core structures are encoded by type-2 PKSs. Type-1 PKSs are “complex” or “modular” PKSs which include assemblies of several large multifunctional proteins carrying, between them, a set of separate active sites for each step of carbon chain assembly and modification. As such, structural diversity occurs in this class from variations in the number and type of active sites in the PKSs. This class of PKSs displays a one-to-one correlation between the number and clustering of active sites in the primary sequence of the PKS and the structure of the polyketide backbone. The second class of PKSs, called Type-2 PKSs, is represented by the synthases for aromatic compounds. Type-2 PKSs have a single set of iteratively used active sites.
Angucycline group antibiotics, which are arranged by a type-2 PKS are structurally characterized by their angular, polyketide-derived benz[a]anthracene-derived backbone (angucyclinone), which is often further decorated with sugar moieties (angucyclines). Angucyclines/angucyclinones form the largest and structurally most diverse sub-group of the multicyclic, aromatic polyketides. Knobler, R. M., Radlwimmer, F. B. and Lane, M. J. Nucleic Acid Res. 20:4553-4557 (1992); Matsumoto, A. and Hanawalt, P. C. Cancer Res. 60:3921-3926 (2000). Yamashita, N., Shin-Ya, K., Furihata, K., Hayakawa, Y. and Seto, H. J. Antibiot. 51: 1105-1108 (1998); Nakashima, T. et al. U.S. Pat. No. 6,030,951. A very interesting set of natural products with respect to their biosyntheses as well as their biological activities derive from this angucycline/angucyclinone group. However, they are not easily recognizable as such, since their polyketide-derived skeleton is rearranged in a series of steps, initiated by oxidative biosynthetic processes. The gilvocarcin-type anticancer antibiotics (Morimoto, M., Okubo, S., Tomita, F. and Marumo, H. J. Antibiot. 34:701-707 (1981); Breiding-Mack, S. and Zeeck, A. J. Antibiot. 40:953-960 (1987); Yamashita, Y. and Nakano, H. Nucleic Acids Res. Symp. Ser. 20:65-67 (1988); Elespuru, R. K. and Gonda, S. K. Science. 223:69-71 (1984)) and the jadomycins (Oyola, R., Arce, R., Alegria, A. E. and Garcia, C. Photophysical properties of gilvocarcins v and m and their binding constant to calf thymus DNA. Photochem. Photobiol. 65:802-810 (1997)) are examples of such ‘rearranged angucyclines’. Both of them, and the kinamycins (Takahashi, K. and Tomita, F. J. Antibiot. 36:1531-1535 (1983)), have in common biosynthetic rearrangement cascades that begin with an oxidative cleavage of the 5,6-bond of an angucyclinone intermediate (FIG. 1).
Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low expression of polyketides in wild-type cells that produce them naturally, there has been considerable interest in finding improved or alternate means to produce polyketide compounds. This interest has resulted in the cloning, analysis, and manipulation by recombinant DNA technology of genes that encode PKS enzymes.
Gilvocarcin-Type Natural Aryl-C-Glycoside Antibiotics
The benzo[d]naphtho[1,2-b]pyran-6-one C-glycoside antibiotics, often referred to as gilvocarcin-type aryl-C-glycosides, were discovered in Japan in the early 1980s. Studies have shown that these molecules are decaketides, and that they originate either from one acetate starter and nine malonate extender units or from one propionate starter and nine malonate extender units, depending on the 8-side chain. The incorporation pattern suggests the key intermediate to be an angucyclinone, such as 2 in FIG. 2., which then rearranges to form the coumarin frame. Krohn, K. and Rohr, J. Angucyclines: Total Syntheses, New Structures, and Biosynthetic Studies of an Emerging New Class of Antibiotics. Topics Curr. Chem. 188, 127-195 (1997); Takahashi, K. & Tomita, F. Gilvocarcins, New Antitumor Antibiotics. 4. Mode of action” J.Antibiot. 35: 1038-1041 (1982); Carter, G. T., Fantini, A. A., James, J. C., Borders, D. B. & White, R. J. Biosynthesis of Ravidomycin. Use of 13C-13C Double Quantum NMR to Follow Precursor Incorporation. Tetrahedron Lett. 25, 255-258 (1984); Carter, G. T., Fantini, A. A., James, J. C., Borders, D. B. & White, R. J. Biosynthesis of Chrysomycins A and B. Origin of the Chromophore. J. Antibiot. 38, 242-248 (1985). Gilvocarcins are biosynthesized by a type-II polyketide synthase (PKS) and the necessary post-PKS tailoring enzymes. Among these, the key enzyme responsible for the tremendous structural change from the suppossed angucyclinone intermediate (e.g., 2 in FIG. 2.) to the unique tetracyclic lactone structure of the gilvocarcins is proposed to be a C—C-bond cleaving oxygenase. Other key post-PKS tailoring steps with respect to important structural features of gilvocarcin V are the oxygenation/dehydration reactions necessary for the formation of the vinyl side chain, and the C-glycosyltransfer step, through which the 6-deoxy-D-fuco-hexofuranose moiety is attached.
This distinct family of antitumor antibiotics shows excellent antitumor activity and remarkably low toxicity, and therefore has remained to be attractive for synthetic organic chemistry as well as for biological activity studies since their discovery. The group consists of the gilvocarcins (syn. toromycins, anandimycins), ravidomycins, the ravidomycin analogues FE35A and B, the chrysomycins (syn. virenomycin, albacarcins; including recent derivatives possessing branched ketofuranose and ketopyranose sugar moieties), and BE-12406 A and B (FIG. 3.). Hirayama, N., Takahashi, K.; Shirahata, K., Ohashi, Y., Sasada, Y. Bull. Chem. Soc. Jap. 54:1338-1342 (1981); Krohn, K. et al. J. Topics Curr. Chem. 188:127-195 (1997); Hosoya, T., Takashiro, E., Matsumoto, T., Suzuki, K. J. Am. Chem. Soc. 116:1004-1015 (1994); Knobler, R. M. et al. Nucleic Acid Res. 20:4553-4557 (1992); Matsumoto, A. et al. Cancer Res. 60:3921-3926 (2000); Yamashita, N. et al. Antibiot. 51:1105-1108 (1998); Nakashima, T. et al. U.S. Pat. No. 6,030,951; Morimoto, M. et al. Antibiot. 34:701-707 (1981).
Gilvocarcin V (GV) (FIG. 4.), the principal product of Streptomyces griseoflavus Gö 3592 and of various other Streptomyces strains, is the most important member of the gilvocarcin-type aryl-C-glycosides, because of its potent bactericidal, virucidal, cytotoxic and antitumor activities. GV is one of the strongest antitumor compounds among these drugs, requiring only low concentrations and maintaining a low in vivo toxicity. The exact molecular mechanisms responsible for the in vivo mode of action of GV are still widely unknown. However, it was found that GV exhibits a strong tendency to intercalate with DNA. Both equilibrium DNA binding and UV light-induced DNA adduct formation was found, causing also topoisomerase II inhibition. Knobler, R. M. et al. Nucleic Acid Res. 20:4553-4557 (1992). The vinyl group is essential for the antitumor activity, since the minor congeners gilvocarcins M and E, in which the vinyl group is replaced by a methyl group and an ethyl group, respectively, are significantly less effective. Yamashita, Y. et al. Nucleic Acids Res. Symp. Ser. 20: 65-67 (1988); Elespuru, R. K. et al. Science. 223:69-71 (1984); Oyola, R. et al. Photochem. Photobiol. 65:802-810 (1997). Photobiological studies showed that the vinyl group undergoes a [2+2] cycloaddition with DNA thymine residues under photoirradiation. Moreover, it was shown recently that Givocarcin V promotes protein-DNA cross-linking when photo-activated by near-UV light, and histone H3, which plays an important role in DNA replication and transcription, was identified as one of the selectively cross-linked proteins (FIG. 5.). This cross-linking with histone H3, believed to be part of the unique molecular mechanisms of the potent antitumor activity of gilvocarcin V, might contribute to the better and more specific activity of GV compared to other intercalating antitumor drugs. Matsumoto, A. et al. Cancer Res., 60:3921-3926 (2000).3b 
The molecular architecture of gilvocarcin V in conjunction with its biological activity makes GV an excellent target for the study of its biosynthesis and the development of novel, improved anticancer, immunosuppressant, antibiotic, antiviral and neuroprotective drugs through combinatorial biosynthesis.